Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA.
Department of Pharmacology and Experimental Neuroscience, College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA.
Nat Protoc. 2019 Aug;14(8):2452-2482. doi: 10.1038/s41596-019-0187-x. Epub 2019 Jul 24.
Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.
收获胚胎从一组女性，微注射试剂胚胎体外和手术转移到另一组女性。虽然乏味，这些方法已经使用了三十多年来创建小鼠模型。我们最近开发了一种方法命名为 GONAD （通过输卵管核酸传递进行基因组编辑），它绕过了这些步骤。GONAD 涉及到将 CRISPR 组件（Cas9 mRNA 和指导 RNA（gRNA））注射到受孕后 1.5 天的输卵管中，然后进行体内电穿孔，将组件递送到原位受精卵中。使用 GONAD，我们证明了可以在小鼠胚胎发育的不同阶段中断和分析靶基因。随后，我们通过将 CRISPR 核糖核蛋白（RNP；Cas9 蛋白或 Cpf1 蛋白和 gRNA）递送到 0.7 天的怀孕小鼠中，开发了改良的 GONAD（i-GONAD），使其适合常规生成敲除和大片段缺失的小鼠模型。当提供单链 DNA（ssDNA）修复模板时，i-GONAD 还可以生成包含多达 1kb 插入物的敲入模型。i-GONAD 提供了其他优势：它不需要输精管切除术的雄性和假孕雌性，用于 i-GONAD 的雌性不被牺牲，并且可以用于其他实验，它可以很容易地在缺乏复杂微注射设备的实验室中采用，并且可以由熟练的小动物手术但缺乏胚胎处理技能的研究人员来实施。在这里，我们提供了建立 i-GONAD 方法的分步协议。该方案需要大约 6 周的时间来生成创始小鼠。
